Intervention by promoting mTORC1 inhibition has emerged as a promising therapeutic strategy to treat aging and aging-related diseases such as cancer. How mTORC1 inhibition contributes to such beneficial outcomes for anti-aging remains unclear. Our long-term goal is to understand how mTORC1-dependent autophagy regulates aging and how the knowledge can be utilized to prevent aging and aging-related diseases. The objective of the current application is to understand how ULK1 and ULK2 (mammalian homologues of Atg1) mediates mTORC1 activity toward the regulation of mitochondrial homeostasis and cellular aging. The central hypothesis is that mTORC1 inhibition activates ULK1 and ULK2 thereby facilitating the turnover of damaged mitochondria, the main source of reactive oxygen species (ROS) that can cause oxidative damages, via mitochondrial autophagy (mitophagy). Our hypothesis has been formulated based on our preliminary data demonstrating protein complexes containing ULK and Atg13. We identified these protein complexes as targets of mTORC1 and the mediators of mTORC1 activity toward the autophagy machinery, defining the long sought-after molecular link between mTORC1 and autophagy. The rationale for the proposed research is that understanding how ULK regulates mitochondrial homeostasis and cellular aging will advance the fundamental knowledge of the role of mTORC1 in the regulation of longevity and assist in the development of molecular targets that are currently lacking to enhance the turnover of defective mitochondria specifically. The hypothesis will be tested by pursing the following three specific aims: 1) Define the role of ULK in mTORC1-mediated oxidative stress response and senescence; 2) Define the mTOR-ULK-mitochondrial pathway; 3) Determine how ULK regulates mitochondrial turnover. Under the first aim, we will analyze the effects of ULK1 and ULK2 depletion on accumulation of damaged mitochondria, oxidative stress response, and cellular senescence. Under the second aim, we will identify ULK phosphorylation sites on Atg13 that regulate the ULK recruitment to damaged mitochondria. Under the third aim, we will identify mitochondrial proteins phosphorylated by ULK1 and ULK2 and characterize their function in mitophagy and mitochondrial dynamics. The research proposed in this application is highly innovative, because it focuses on a previously unexplored pathway that fills in the current existing gap that links mTORC1 with the regulation of mitochondrial homeostasis. The proposed research is significant, because it will transform understanding of mTORC1 function in aging to an unprecedented level of detail. This is the essential first step in research that is expected to enable development of strategies that specifically manipulate the mTORC1-ULK-mitochondrial pathway to suppress cellular aging and cancer.